Iranian Journal of Pharmaceutical Research (2012), 11 (2): 611-620 Copyright © 2012 by School of Pharmacy Received: December 2010 Shaheed Beheshti University of Medical Sciences and Health Services Accepted: August 2011

Original Article

Antinociceptive and Smooth Muscle Relaxant Activity of tiglium L Seed: An In-vitro and In-vivo Study

Zhen Liua, Wenyuan Gaoa*, Jingze Zhanga and Jing Hua,b

aSchool of Pharmaceutical Science and Technology, Tianjin University, Tianjin 300072, China. bSchool of Chinese Medicine, Tianjin University of TCM, Tianjin 300193, China.

Abstract

The seed of Croton tiglium L. (SCT) is a well known folk medicine. In China, it has used to treat gastrointestinal disorders, intestinal inflammation, rheumatism, and so on. Previous studies established its purgative and inflammation properties. In addition, the effects of essential oil of SCT on intestinal transit and gastrointestinal tract has been studied. In the present study, we evaluated the antinociceptive effect of SCT through the writhing test in mice, investigated the effects of it on spontaneous smooth muscle contractions of isolated rabbit jejunum and examined the in-vitro results through the in-vivo small intestine propulsion. We further investigated the possible compounds using HPLC-MS, and six compounds were tentatively identified as phorbol esters. Furthermore, the possible fragmentation pathways of phorbol esters were proposed, and we also detected the possible compounds in the active parts.

Keywords: Croton tiglium L; Intestinal propulsion; Smooth muscle; Rabbit jejunum; Antinociceptive; Phorbol esters.

Introduction guinea pig colonic smooth muscle cell has been studied, which regulates the gastrointestinal The genus Croton belongs to the family transit in mice, and affects the inflammatory and . The seed of Croton tiglium L. immunological milieu (2, 5). In the previous (SCT) is well known as Ba-Dou in China. It has study, we reported the effect of croton oil on been used as a traditional medicine for many spontaneous smooth muscle contractions in applications such as constipation, a purgative, and isolated rabbit jejunum and the underlying treating dyspepsia and dysenteria. The Chinese mechanisms (6). From the leaves of C. tiglium, had written records in the second century B.C. a pyrazine derivative crotonine was isolated and for using it to treat the gastrointestinal disorders, showed significance analgesic effects (7). intestinal inflammation, rheumatism, headache, Some tigliane phorbol esters have been peptic ulcer and visceral pain (1-4). Croton oil, isolated from C. tiglium previously. Among the essential oil of SCT, as the effective part, Croton , only C. tiglium has been has been reported to have purgative, analgesic, extensively studied as the source of phorbol antimicrobial, and inflammatory properties (1, derivatives (8, 9). Phorbol esters have been 3). Besides, the direct effect of Croton oil on shown to be responsible for eliciting a remarkable range of biochemical effects except * Corresponding author: tumor promoting (10, 11), such as skin irritant E-mail: [email protected] effects (12), platelet aggregation (13), and cell Liu Z et al. / IJPR (2012), 11 (2): 611-620 differentiation (14). Although the ability of these induced writhing in mice, and investigated the compounds to promote tumors presents the active fraction of SCT on spontaneous smooth potential limitation of their utility, it should be muscle contraction in isolated rabbit jejunum. stressed that there are many phorbol esters that By the results of the muscle contraction, we exert the profound beneficial biological effects tested the effect of SCT on intestinal propulsion without tumorigenesis. 12-O-tigloylphorbol-13- in mice using the charcoal method. The decanoate isolated from croton oil demonstrated effective compounds of SCT and the possible antileukemia activity against the P-388 leukemia fragmentation of phorbol esters are also in mice (15). Eight phorbol esters isolated from described in this assay. the C. tiglium have the ability to inhibit an HIV- induced cytopathic effect on MT-4 cells (16). Experimental The most investigated activity of the phorbol esters is their binding and activation of protein materials and animals kinase C (PKC), which plays a critical role in The seed of C. tiglium was provided by Tianjin signal transduction pathway and regulates the Lerentang Pharmaceutical Factory (Tianjin, cell growth and differentiation (17, 18). PKC China) and identified by Professor Wenyuan isoforms are distributed in the small intestine Gao from School of Pharmaceutical Science and involved in modifying the functions of and Technology, Tianjin University, China. The intestinal muscle including the generation of voucher specimen (voucher No. BD070701) are slow, sustained contraction of smooth muscle available in the herbarium of Research Center of cells through Ca2+ influx, as shown through the Tianjin Zhongxin Pharmaceuticals. experiments using phorbol esters and isozyme- Adult male and female New Zealand specific blockade (19, 20). Our previous study white rabbit (2.0-2.5 kg) were obtained from proved quite consistent with this (6). Laboratory Animal Center of Peking University The commonly used models of analgesia (Beijing, China). All animals were housed at the contain thermal stimulation, electrical Experimental Animal Center of Tianjin Medical stimulating method, mechanical irritation and University (Tianjin, China) and kept under chemical stimulus. With different models, there standard environmental conditions. Animals had can be differences in the analgesic effects of free access to water, but food was withdrawn 24 selected drugs; some trends can be identified h before the experiments. KM mice (18-22 g) (21-22). The writhing test is an experimental were housed in plastic cages, with food and tap model used for the screening of drugs with water available ad libitum, in the colony room. analgesic activity, based on the irritation caused The animals submitted to oral administration of after the intraperitoneal injection of 0.6% acetic the extract or drugs were fasted for 24 h. acid. This injection can produce the peritoneal Animals’ experiments were performed with inflammation characterized through the the approval of the Institutional Animals Care contraction of abdominal muscles accompanying and Use Committee of China, and institutional an extension of the forelimbs and elongation of guidelines for animal welfare and experimental the body. This writhing response is considered conduct were followed. to be a visceral inflammatory pain model, and in this way, this acid causes the release of algesic Reagents and chemicals mediators such as bradykinin, prostaglandins, HPLC-grade acetonitrile was from histamine and 5-hydroxytryptamine. Merck (Darmstadt, Germany). Acetylcholine Additionally, although this test was a nonspecific (Ach), Hexamethonium, Methoctramine, model (e.g. anticholinergic and antihistaminic and 4-Diphenylacetoxy-N-methylpiperiding and other agents show activity in this test), it is methiodide (4-DAMP) were purchased from widely used for analgesic screening and involves Sigma (St. Louis, MO, USA ) and Verapamil local peritoneal receptors (23). Hydrochloride Injection was obtained from So in the present study, we examined the Hefeng Co., Ltd. (Shanghai, China). Atropine antinociceptive effects of SCT in acetic acid- sulphate injection and Noradrenaline Bitartrate

612 Antinociceptive and Smooth Muscle Relaxant Activity Croton tiglium

Table 1. Possible compounds from SCTp and SCTe by HPLC-ESI(+)/MSn.

M+H]+ or[ NO Compound Molecular formula +]Fragment ions of [M+H]+ or [M+Na [M+Na]+ m/z

1 Deoxyphorbol acetate methylbutanoate C27H38O7 475.1 265.1 ,293.0 ,277.0 ,311.0 ,355.3

2 Phorbol acetate methylbutenoate C27H36O8 489.2 265.1 ,293.0 ,311.2 ,389.3 ,429.3

3 Deoxyphorbol acetate methylbutenoate C27H36O7 473.0 265.1 ,293.1 ,311.2 ,391.0

4 Phorbol methylbutanoate isobutyrate C29H42O8 519.6 265.1 ,293.0 ,311.0

5 Phorbol decanoate acetate C32H48O8 583.3 265.1 ,269.0 ,293.1 ,311.1 ,501.8

6 Phorbol acetate butyrate C26H36O8 499.1 265.0 ,293.1 ,311.2 ,417.3 were supplied by Jinhui amino Co., Ltd. (Tianjin, blow on the head. After a laparotomy incision, a China). Other chemicals were of the highest portion of the jejunum was removed and placed grade available. in an oxygenated Tyrode’s solution (composition

in mM: NaCl 136.9, CaCl2 1.8, KCl 2.7, MgCl2

Extraction of the seed of C. tiglium 1.1, NaHCO3 11.9, NaH2PO4 0.4, and glucose SCT (2 Kg) was extracted with methanol 5.6, pH = 7.4). Respective five segments of (10L × 3) under reflux for 3 h. The methanol jejunum 2 cm in length were mounted in a 10 mL extracts (SCTm) were combined and evaporated organ bath containing Tyrode’s solution that was under reduced pressure in a rotary evaporator bubbled with a 95% O2 and 5% CO2 gas mixture to give an oily residue (250 g), with a yield of and the temperature was held at 37°С (6). 12.5%. The residue was suspended in aqueous and extracted with petroleum ether, ethyl acetate Contractile activity of smooth muscle and normal butanol. The extracted solutions Each segment was allowed to equilibrate in the were respectively evaporated under reduced bath for 50 min to obtain a regular spontaneous pressure to give P.E. parts (SCTp) (167 g), activity. An initial load of 1 g was applied to each EtOAc parts (SCTe) (8 g), n-BuOH parts (44 g) of the tissue and was kept constantly throughout and H2O fraction (8 g). Croton oil was the same the experiment. The muscle tension, measured as previously reported (6). with a force transducer (Model JH-2, Beijing, China), was displayed on a multichannel recorder Writhing test (HV-4, Taimeng, China) and monitored with a This test was done using the method described Biology BL-410 computer. Then, the following by Koster et al. (25). Seventy male and female experiments were performed. KM mice were used in this experiment and they Croton oil, SCTm, SCTp and SCTe were were divided equally into ten groups. Mice were separately prepared as 40 mg/mL stock solution pretreated as follows: Group 1 (Control group), in 0.5% tween-80 and additional dilutions were water solution (10 mL/Kg, p.o.); Group (2-7), made with distilled water. A single concentration SCTm (25, 50, 100, 200, 250, 300 mg/Kg, p.o.); of them (20, 60, 80, 100, 200 μg/mL) was Group 8, SCTp (20 mg/Kg, p.o.); Group 9, SCTe added to the organ bath., n-BuOH parts and

(20 mg/Kg, p.o.); Group10, Aspirin (100 mg/Kg, H2O fraction were prepared as 0.5 g/mL stock p.o.). All substances were administered 60 min solution in 0.5% tween-80 and additional before the intraperitoneal acetic acid injection dilutions were made with distilled water. The (0.6%, 0.1 mL/10 g, IP) and the number of end concentrations were 0.1, 0.5, 1.0, 2.0 and 4.0 writhes was counted for the following 30 min. mg/mL. In this experiment, each dose that was All the doses of the different parts were based on designed according to the prepared experiment the data showed in the previous paper (6). that displayed a turning point used six tissue preparations. Tissue preparation The average peak intensity, tension and Thirty-six rabbits were sacrificed using a frequency of contractions occurred before (5 min)

613 Liu Z et al. / IJPR (2012), 11 (2): 611-620

Table 2. Effects of SCT extract on acetic acid-induced abdominal writhing in mice. Acetic acid-induced writhing test Treatment Dose (mg/Kg) Number of writhes (30 min) Inhibition (%) Vehicle (p.o.) - 24.5± - 25 20.5± 16.3% 50 17.0± 30.6% 100 13.3± 45.7% SCTm (p.o.) 200 21.7± 11.4% 250 23.3± 1.2% 300 23.8± 2.9% SCTe (p.o.) 20 15.5± 36.7% SCTp (p.o.) 20 15.2± 38.0% Aspirin (p.o.) 100 8.1± 80.0% Values represent the mean ± SEM of 7 mice.* Significantly different from control group: p < 0.05.** Significantly different from control group: p < 0.01. and after (5 min) administration of each drug were HPLC-MS and ESI-MSn of P.E. parts and determined. Relative changes of drug-induced EtOAc parts of C. tiglium contractile responses to the basal levels (before the SCTp and SCTe were analyzed through treatment of drugs) were calculated as percentage. high-pressure liquid chromatograms (HPLC)- mass spectrometer (MS). Briefly, the extracts Small intestinal propulsion were analyzed via HPLC-mass spectrometer The effect of SCT on intestinal propulsion using HPLC (Agilent technologies 1200 series in KM mice was tested using the charcoal Diode Array detector) with an ion-trap ESI- method (26). Fifty male and female KM mice mass spectrometer. Samples were injected into were fasted for 12 h but allowed free access to a Kromasil RP-C18 column (4 mm×250 mm). water. The animals were randomly allotted into The column was equilibrated in water (solution five groups of ten animals per group. Group 1 A) and elution of the components was achieved was administered with distilled water (10 mL/ by increasing the concentration of solution B Kg, p.o.) using orogastric cannula. Group 2 was (100% acetonitrile) from 5 to 95% in 60 min at a pretreated with C. tiglium (200 mg/Kg, p.o.). flow rate of 0.8 mL/min. The molecular masses Group 3 was pretreated with SCTm (50 mg/ of the peaks were determined from electro-spray Kg, p.o.) while group 4 and 5 received SCTp ionization mass spectra using multiply-charged (20 mg/Kg, p.o.) and SCTe (10 mg/Kg, p.o.), ion profile. respectively. Thirty minutes after the treatment The Agilent HPLC-MS system contains a with the extract, each mouse was administered survey or auto-sampling system, interfaced to with 0.2 mL of standard charcoal meal (5% an ion-trap mass spectrometer via an electro- activated charcoal suspended in 10% gum spray ion source. Source setting used for analysis acacia) orally. The mice were killed 30 min the extracts were: nebulizer gas flow, 30.00 psi; later through cervical dislocation, and the small dry gas flow, 8.00 L/min; capillary temperature, intestine was rapidly dissected out and placed on 350°C; Nitrogen (> 99.99%) and He (> 99.99%) a clean surface. The small intestine was carefully were used as sheath and damping gas, respectively. inspected and the distance traversed using the The full scan of ions ranging from m/z 100 to charcoal meal from the pylorus was measured 1,000 in the positive ion mode was carried out. for both the control and treated groups. For each The fragment ions were obtained for both MS2 group, the results were expressed as percentage and MS3 experiments. Analyses were conducted of the distance traveled from the pylorus to the at ambient temperature and the data were operated caecum (27). on the Xcalibur software.

614 Antinociceptive and Smooth Muscle Relaxant Activity Croton tiglium

Table 3. Effects of SCT extracts on small intestine propulsion in mice ( x ± s, n = 10). Treatment Dose (mg/kg) Transversed (%) Control - 57.94 ± 10.82 SCT 200 75.06 ± 10.53 ** SCTm 50 77.62 ± 12.98 ** SCTp 20 83.23 ± 10.72 ** SCTe 10 100.00 ± 0.00 **

* Significantly different from control group: p < 0.05.** Significantly different from control group: p < 0.01.

Statistical analysis amplitude and tension of muscle contractions Data were expressed as mean ± SEM with n and at high concentration, it decreased the denoting the number of tested tissue preparations. intensity and tension of muscle contractions. SPSS 15.0 (for Windows) was used to analyze In addition, they suppressed the frequency the data. Student’s t-test was used to analyze of muscle contractions in a concentration- and compare the results between the groups dependent manner. At the concentrations of 40 while a one-way ANOVA was used to compare μg/mL, SCTe reached the maximum, whereas, the results among the groups. Differences were SCTp and SCTm reached the maximum at the considered statistically significant if p < 0.05. concentrations of 60 μg/mL and 80 μg/mL

(Figure 1). However, n-BuOH parts and H2O Results and Discussion fraction decreased this effect.

The result of writhing test in mice Effect on small intestine propulsion In the writhing test in mice, SCTm at doses of For testing the results of SCT in muscle 25, 50 and 100 mg/Kg, inhibited the frequency- contraction in-vitro, we examined the effect induced abdominal constrictions with acetic acid on small intestinal propulsion in-vivo. In in a dose-dependent manner and results were control animals, at 30 min after the intragastric statistically significant (Table 2). The results administration, the charcoal meal transversed obtained for SCTm, SCTp and SCTe were 57.94% of the total length of the small intestine similarly and all showed weak analgesic effect (Table 3). SCT, SCTm, SCTp and SCTe when compared to aspirin (positive control, 100 promoted small intestinal transit progressively mg/Kg, 80.0%). The oral pre-treatment of mice (75.06-100.00%, p < 0.01). This result consisted with SCTm (100 mg/Kg), SCTp (20 mg/Kg) and with the result in-vitro testing. SCTe (20 mg/Kg) resulted in a 45.7%, 38.0% and 36.7% inhibition of the abdominal writhing, Possible compounds and fragmentation respectively. pathways of phorbol esters of SCTp and SCTe Six compounds speculated in SCTp and Effects of the different parts of C. tiglium on SCTe were summarized in Table 1 by HPLC- spontaneous smooth muscle contractions ESI(+)-MS. From the fragment ions of phorbol The method used in the experiment is the esters and the reported (28), as shown in Figure same as previously reported (6). We observed 2, two fragmentation pathways were proposed. that the action of SCTm was concord with The main fragments in the compounds were that of Croton oil, but with a better effect m/z 311(C20O3H23), 293 and 265, which was in muscle contraction. SCTp and SCTe loss three molecules of water or organic acid increased the activity of muscle contraction, (ROOH). In Figure 3, the fragmentation and the tendency of the effect was the same pathways of compound deoxyphorbol as SCTm, which at the low concentrations, acetate methylbutanoate and phorbol acetate concentration-dependency increased the methylbutenoate were shown.

615 Liu Z et al. / IJPR (2012), 11 (2): 611-620

8080 160 ** SCTm 160 6060 SCTp 140 ** SCTe 140 ** SCTm 4040 ** SCTp 120120 * ** SCTe 2020 ** ** 100100 0 0 ** 8080 ** * -20 60 -20 ** 60 **

Intensity (%) ** ** I nt ensi t y（%） Tension (%) Tension

-40-40 ** Tensi on（%） 4040 ** ** ** ** * * -60 2020 * -60 ** ** -80 00 -80 ** * 0.000.00 0.04 0.08 0.120.12 0.16 0.20 0.00 0.04 0.080.08 0.120.12 0.160.16 0.20 Concentration (mg/mL) Concentration (mg/mL) （ A） Concent r at i on（mg/mL） （ B） Concent r at i on（mg/mL）

-35-35 SCTm -30-30 SCTp SCTe

-25-25 * ** * -20-20

-15-15

Fr equnency（%） * Frequnency (%) -10-10 * -5-5

00

0.00 0.040.04 0.08 0.12 0.160.16 0.20 （ C） Dose (mg/mL) Dose（mg/mL）

Figure 1. Representative intensity (A) and tension (B) or Frequency (C) on the smooth muscle contractions in isolated rabbit jejunum induced by SCTm, SCTp and SCTe with five different doses (0-0.2 mg/mL). The dates were measured by isometric force transducers before (5 min) and after (5 min) treatment of each parts. Each point represents mean ± SEM. of six tissues (n = 6). * p < 0.05 **, p < 0.01 compared to the corresponding values of basal contractility.

Conclusion releasing endogenous mediators that stimulate the nociceptive neurons (29). In the present In the present study, we evaluated the writhing test, there was a little reduction in antinociceptive effect of SCT through the writhing for the groups treated with SCTm, writhing test in mice, investigated the effects of SCTp and SCTe; the inhibition were lower than it on spontaneous smooth muscle contractions of 50% (Table 2). Although an analgesic pyrazine isolated rabbit jejunum, and examined the results derivative isolated from the leaves of C. tiglium in-vitro through the small intestine propulsion in- could remarkably inhibit the acetic acid- vivo and the possible compounds were detected induced abdominal writhing in mice (7). From in the active parts. the results, SCT may decrease the abdominal Acetic acid-induced writhing is a standard, pain in some traditional Chinese prescription, simple, and sensitive test for measuring analgesia such as Wei-Chang-An-Wan (30). However, in and has long been used as a screening tool for this model, it is postulated that the abdominal the assessment of analgesic or anti-inflammatory constriction response is induced through local properties of plant extracts and natural products. peritoneal receptor activation and involves It has been suggested that acetic acid acts by prostanoids mediator (31). Additionally, an

616 Antinociceptive and Smooth Muscle Relaxant Activity Croton tiglium

OR1 OR2 OR2

- OR1-H - OR2-H OH OH O OH O OH OH OH OH O OH + OH C20H25O4 Ⅰ m/z 329.1753 - H20

- H O -CO 2 OH O O OH OH OH + C19H21O + + C20H21O2 C20H23O3 m/z 265.1592 m/z 293.1542 m/z 311.1647

OR1

- OR1-H - R2-H OH OH O OH O OH OR2 OH O

O OH + OR2 C20H25O4 Ⅱ m/z 329.1753 - H20

- H 0 -CO 2 OH O O OH OH OH + C19H21O + + C20H21O2 C20H23O3 m/z 265.1592 m/z 293.1968 m/z 311.1804

-Deoxy phorbol (׀׀) ;phorbol 12,13-diesters (׀) :Figure 2. Proposed fragmentation pathways and characteristic ions of Phorbol esters 13,20-diesters. important disadvantage of this model is that the that Croton oil possessed spasmogenic and other classes of drugs can reveal the effect, such spasmolytic properties and the regulatory effects as adrenergic antagonists and muscle relaxants, of Croton oil on gastrointestinal motility were favoring possible false positive results (21). Due mediated via the activation of M3 muscarinic to these reasons, the results from the writhing receptor and Ca2+ influx through L-type2+ Ca test should be for further investigation with other channel (6). In this experiment, the effect of Croton tests, such as hot plate test and formalin test. oil and SCTm on smooth muscle contractions In our previous work, it was demonstrated was compared. Through further investigated,

617 Liu Z et al. / IJPR (2012), 11 (2): 611-620

OAc

-HO2C5H9 -AcOH + OH H -H2O -H2O OAc + O CH O CH+ m/z 355 m/z 277 + H H+ OH

HO -C H OH O O2C5H9 5 9 -H O + OH 2 C27H39O7 -AcOH -H2O O O m/z 475 O O Compound 1 m/z 311 m/z 293

-CO

H+

O

+ m/z 265 O H

O H+ O O -CH3COOH O OH O O OH OH + C25H33O6 -C5H8O2 -H2O OH m/z 429 O O OH OH O H+ + + H C27H37O8 m/z 489 -C5H8O2 -CH3COOH -H2O

Compound 2 OH O OH OH O OH OH + C20H23O3 + C22H29O6 m/z 311 m/z 389 -H20 H+ H+ - CO

OH O + OH C19H21O2 C H O + m/z 265 20 21 2 m/z 293

Figure 3. Proposed fragmentation pathways and characteristic ions of compound 1 and compound 2. the different parts of SCTm and both of SCTp a biphasic action. SCTp and SCTe had the same and SCTe parts showed contract intestinal tissue, effect on gastrointestinal motility as Croton oil, and conversely, n-BuOH parts and H2O fraction which possessed spasmogenic properties at low had the action of relaxing intestinal tissue. These concentration (0-0.06 or 0-0.04 mg/mL) and findings suggested that C. tiglium itself possess spasmolytic properties at high concentration

618 Antinociceptive and Smooth Muscle Relaxant Activity Croton tiglium

(0.06-0.1 or 0.04-0.06 mg/mL). In the in-vivo study. The results demonstrated that SCTp study, SCTm at dose of 50, 100 and 200 mg/ and SCTe could contract the smooth muscle Kg increased the intestine propulsion in a dose- and six phorbol esters were detected from dependent manner, however, at dose of 200, 400 them. However, the chemical compounds and and 800 mg/Kg, the inhibitions of the intestinal the exact mechanisms of contraction must be propulsion were lower (data was not shown). further investigated. SCTp and SCTe exhibited higher increasing in the intestinal propulsion (Table 3). Both of Acknowledgment in-vitro and in-vivo results suggested that C. tiglium possessed spasmogenic and spasmolytic The work was supported by a grant from properties at different doses, and SCTp and Modernization of Traditional Chinese Herbs of SCTe were the active parts. So in the following Tianjin, China (No. 05ZHGCGX01000). work, we proposed the possible compounds in SCTp and SCTe by LC-MS. References It was reported that phorbol esters were (1) Qiu HX. Flora of China. Science Press, Beijing (1996) the main active compounds in C. tiglium, and 133. they were known as the activator of protein (2) Wang X, Lan M, Wu HP, Shi YQ, Lu J, Ding J, Wu kinase C (PKC). PKC activation mediated KC, Jin JP and Fan DM. Direct effect of croton oil on various signaling pathways critical for the intestinal epithelial cells and colonic smooth muscle formation, regulation, and maintenance of the cells. World J. Gastroenterol. (2002) 8: 103-107. (3) Tsai JC, Tsai S and Chang WC. Effect of ethanol gastrointestinal tract (32). Phorbol esters could extracts of three Chinese medicinal with induce rapid, sustained contraction in smooth laxative properties on ion transport of the rat intestinal muscle cells isolated from guinea pig intestine epithelia. Biol. Pharm. Bull. (2004) 27: 162-165. and the contraction of phorbol was related (4) Morimura K. The role of special group article in ancient to Ca2+ (19, 20). The mechanism of Croton Chinese medical prescription. Historia Scientiarum oil induced contraction was mediated via (Tokyo) (2003) 13: 1-12. 2 2+ (5) Wang X, Zhang FM, Liu ZX, Feng HZ, Yu ZB, Lu Ca +influx through L-type Ca channel (6). So, YY, Zhai HH, Bai FH, Shi YQ, Lan M, Jin JP and Fan the active compounds for C. tiglium induced DM. Effects of essential oil from Croton tiglium L. on muscle contraction in isolated rabbit jejunum intestinal transit in mice. J. Ethnopharmacol. (2008) maybe phorbol esters. 117: 102-107. (6) Hu J, Gao WY, Gao Y, Ling NS, Huang LQ and Liu By HPLC-ESI-MS/MS, six compounds 2+ CX. M3 muscarinic receptor- and Ca influx-mediated (Table 1) were speculated from the SCTp and muscle contractions induced by croton oil in isolated SCTe of C. tiglium. In Figure 2, we proposed rabbit jejunum. J. Ethnopharmacol. (2010) 129: 377- the possible fragmentation pathways of phorbol 380. esters. The characteristic fragment ions were (7) Wu XA, Zhao YM and Yu NJ. A novel analgesic m/z 311, 293 and 265. In LC-MS, side chains of pyrazine derivative from the leaves of Croton tiglium phorbol esters such as fatty acid and H O could L. J. Asian Nat. Prod. Res. (2007) 9: 437-441. 2 (8) Hecker E. Phorbol esters from croton oil chemical be eliminated to give a stable fragment ion. To nature and biological activities. Naturwissenschaften obtain a similarly stable fragment ion, only two (1967) 54: 282-284. esters/hydroxyl groups of deoxyphorbol esters (9) Baird WM and Boutwell RK. Tumor-promoting were eliminated as free acid or water. The activity of phorbol and four diesters of phorbol in fragment ions of phorbol esters in the MS-MS mouse skin. Cancer Res. (1971) 31: 1074-1079. (10) Berenblum T and Shubik P. The role of croton oil mode were m/z 311→293, and a characteristic applications associated with a single painting of a elimination of 28 u (-CO) from the fragment carcinogen in tumor induction of the mouse’s skin. Br. m/z 293 could be observed, thus, m/z 293 J. Cancer (1947) 1: 379-382. →265 could be detected (28). (11) Matsukura N, Kawachi T, Sano T, Sasajima K In conclusion, the effects of muscle and Sugimura T. Promoting action of croton oil contraction and analgesic of SCT extracts both on gastrocarcinogenesis by N-methyl-N’-nitro-N- nitrosoguanidine in rats. J. Can. Res. Clin. (1979) 93: in-vitro and in-vivo were investigated and the 323-327. compounds were analyzed in this experimental (12) Schmidt RJ and Evans FJ. Skin irritant effects of esters

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